Targeted Drug Delivery and Treatment of Endoparasites with

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Targeted drug delivery and treatment of endoparasites with biocompatible particles of pH responsive structure Patrick D. Mathews, Ana Carolina Monge Fernandes Patta, Joao Vitor Gonçalves, Gabriella dos Santos Gama, Irene Teresinha Santos Garcia, and Omar Mertins Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b01630 • Publication Date (Web): 28 Dec 2017 Downloaded from http://pubs.acs.org on January 2, 2018

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Targeted drug delivery and treatment of endoparasites with biocompatible particles of pH responsive structure Patrick D. Mathewsa*, Ana C. M. Fernandes Pattaa, Joao V. Gonçalvesa, Gabriella dos Santos Gamaa, Irene T. S. Garciab, Omar Mertinsa* a

Department of Biophysics, Paulista School of Medicine, Federal University of Sao Paulo, Sao

Paulo 04023-062, Brazil b

Department of Physical-Chemistry, Institute of Chemistry, Federal University of Rio Grande

do Sul, Porto Alegre 91501-970, Brazil

*Correspondence: [email protected] (P.D.M.) [email protected] (O.M.) Tel.: +55 11 5576 2338 Fax: +55 11 5571 5780

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ABSTRACT

Biomaterials conceived for vectorization of bioactives are currently considered for biomedical, biological and environmental applications. We have produced a pH sensitive biomaterial composed of natural source alginate and chitosan polysaccharides for application as drug delivery system via oral administration. The composite particles preparation was in situ monitored by means of isothermal titration calorimetry. The strong interaction established between the macromolecules during particles assembling lead to 0.60 alginate/chitosan effective binding sites, with intense exothermic effect and negative enthalpy variation in the order of thousand kcal/mol. In presence of model drugs mebendazole and ivermectin, of relative small and large structure respectively, mebendazole reduced 27% the amount of chitosan monomers available to interact with alginate, which was not observed for ivermectin. Despite, a state of intense negative Gibbs energy and large entropic decrease was achieved evidencing that formation of particles is thermodynamically driven and favored. Small angle Xray scattering further evidenced similar surface aspect independently of drugs absence or presence. The physical responses of the particles to pH variation comprise partial hydration, swelling and predominance of positive surface charge in strong acid medium, while ionization followed by deprotonation leads to compaction and charge reversal than new swelling respectively in mild and slight acid mediums. In vivo performance was evaluated in treatment of endoparasites in Corydoras fish. Systematically in a daily base oral administration, particles reduced significantly the infections in 15 days of treatment. The experiments evidenced that utilization of particles granted and boosted the action of the anti-parasitic drugs, leading to substantial reduction or elimination of infection. Hence, the pH responsive particles represent a biomaterial of prominent characteristics liable to development of target oral drug delivery.

KEYWORDS: Composite bioparticles; Isothermal titration calorimetry; SAXS; Parasitosis; In vivo application; Prevalence.


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1. INTRODUCTION

Efficient in vivo performance of drug delivery systems has received increasing regard with the development of micro and nano particles of specific structural characteristics, allowing responsive behavior in determined environment conditions.1-4 Actually, the target drug delivery relies on the concept where a specific drug carrier incorporates the drug protecting it from undesired degradation, “safely” carries the same throughout deleterious route of administration, achieving the target site and there ideally delivers the drug in a sustained release in order to warranty ideal pharmacological action. Afterwards, an easy removal of the now “empty” carrier is desired, avoiding cumulative disorders or side effects, which may be attained with the use of biocompatible and biodegradable materials.5,6 Therefore, the development of biomaterials of responsive structural behavior depending on physico-chemical characteristics of the whole route of administration plus the target site is a matter of actual scrutiny. In development of a drug delivery biomaterial one primarily ought to consider the route of administration, i.e., intravenous, oral, ocular, transdermal and so on. The oral administration is assumed as better alternative to parenteral administration considering principally the convenience and increased compliance to patients.7 Nevertheless, many drugs are degraded or loose the activity during route of administration, hence before achieving the aimed target of desired treatment site. Especially to the oral route, one important and no neglecting feature to consider is the pH variation.8 Indeed, the same may vary from neutral to strong acid than alkaline, if considering buccal, stomach and intestinal tract. In profit of the pH variation, specific pH-responsive biomaterials are under development, denoting promising and improved therapeutic action of transported drugs. For instance, nanoparticles made with acrylic based polymers respond to acid pH of stomach by carboxyl protonation.9 Later through gastrointestinal tract when achieving higher pH, hydrogen bond breakage and ionization of the same carboxyl leads to swelling of particles, hence promoting release of encapsulated insulin. Han et al.10 developed a more specific pH sensitive capsule to target colon and release theophylline. The in vivo study in rats demonstrated that the capsule kept intact from stomach to small intestine while disintegration occurred in the proximal colon, thus optimizing the target drug delivery. Despite all the progress over the last years, oral delivery pH-responsive materials require further improvement, especially concerning increase of delivery specificity to the site of drug expected action,11 thus improving effectives of treatments. Within this concept, all stages involved in design of the drug delivery system have to be carefully unveiled aiming the 3 ACS Paragon Plus Environment


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production of a multifunctional device of known pH-responsive characteristics and prominent to specific site drug targeting. Besides, in vivo applications require safety of the drug carrier, demanding the use of materials of low immunogenic toxicity, preferred biocompatibility and easy biodegradation or elimination after achieving the target and the expected activity. In these senses, the biomacromolecules alginate and chitosan meet such good requirements, since both natural source polysaccharides are known as biocompatible and biodegradable. Chitosan has been studied in diverse drug delivery systems ranging from oral, nasal, intravenous or transdermal administration.12,13 Alginate has received attention due to excellent gel forming property, inputting development of gel based drug delivery besides wound dressings.14,15 The combination of both polysaccharides has shown further promising applications in the biomedical field16-18 and it relies on the opposite charge profile of the macromolecules in solution, with pKa 6.0 for chitosan and 3.38 (mannuronate residues) and 3.65 (guluronate residues) for alginate, which allows molecular interaction that may be controlled in specific experimental conditions, leading the assembling of drug carriers of defined characteristics. Alginate/chitosan particles may be produced by complex coacervation, where the opposite charges of the biopolymers in solution promote crosslinking and assembling of particles.19-21 Nevertheless, the strength of electrostatic interaction, which provides the final structures and properties, has fairly been described in the assembling process of such drug carriers production. Within these challenges, in the present article a detailed physico-chemical study of a drug delivery particle is primarily reported. Composite alginate-chitosan particles have been produced, however the interaction between the biomolecules was in situ thermodynamically monitored during the assembling in preparation. Beyond, two hydrophobic model drugs, namely mebendazole and ivermectin, of relative small and large molecular structure respectively, were incorporated in the particles and the influence and location of both was evaluated. Both drugs are well known as anthelmintic drugs of negligible water solubility, but predicted partition coefficients (cLogP) 2.95 and 5.83 respectively, characterizing high interaction with hydrophobic media.22 With these characteristics, the use of drug carriers becomes important to allow and promote delivery to specific sites of action. The pH-responsive behavior of the particles has been monitored in a large range of pH variation concerning production and application conditions in order to determine the related structural characteristics. The target drug delivery, by applying the composite particles via oral administration, was further studied in vivo in treatment of parasites in Corydoras fish highly infected in whole gastrointestinal tract. The efficacy of the particle was hence tested with vectorization of the 4 ACS Paragon Plus Environment

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anti-parasitic drugs and effectiveness of treatment. Thereby, the development of a pH responsive drug delivery system of technologic appeal and specific applicability in oral administration is here described.

2. MATERIALS AND METHODS

2.1. Materials and animals

Chitosan ChitoClear® 43000 (Pandalus borealis; batch: TM4545) was provided by Primex (Iceland), with 95% degree of deacetylation (DDA), average molecular weight Mw = 130 kDa (corresponding to 797 repeat monomers per molecule) and low viscosity (< 20 cP). In order to eliminate traces of impurities,23 chitosan was dissolved at 5 mg/mL in acetic acid solution (50 mM, pH 3.0). The solution was stirred 24 h, 1500 rpm, at room temperature, than filtered through cellulose acetate Millipore membrane (0.80 µm pore size). After, chitosan was precipitated by addition of NaOH (10% solution until pH 8.5), then filtered (G2 sintered glass) and washed with purified water (Milli-Q) until a conductivity of 4.5 mS in washing water was achieved. Then, it was further washed with water/ethanol solutions (30:70, 20:80, 10:90 and 0:100, v/v), dried and kept in a silica gel desiccator under vacuum. Sodium alginate (from brown algae Macrocystis pyrifera) was from Sigma-Aldrich (St. Louis, MO, USA) with 61% mannuronic acid and 39% guluronic acid, average Mw = 200 kDa (1143 uronic acid units) and low viscosity (5-40 cP). Chitosan stock solution was prepared by overnight stirring in acetate buffer24 (80 mM, pH 4.50 ± 0.02) at 1 mg/mL and diluted when required. Mebendazole (5benzoyl-2-benzimidazolecarbamic acid methyl ester; 98%) was from Sigma-Aldrich and ivermectin Ivermectan® (22,23-dihydroavermectin) for veterinary use was provided by Uzinas Chimicas (Jaboticabal, Brazil). All reagents were of analytical grade and solutions were prepared using deionized water from Milli-Q Millipore system with a total organic carbon value of less than 15 ppb and a resistivity of 18 MΩ cm. Fish were adult Corydoras agassizii collected from Amazon basin at Rio Negro River, municipality of Santa Isabel do Rio Negro (0° 24′ 50′′ S, 65° 01′ 08′′ W), Amazonas State, Brazil, in November 2016, with 4.83 g average weight, measuring between 5 and 6 cm length, and transported to the laboratory under appropriate conditions of temperature and water aeration according to Brazilian law.25 Upon arrival, fish were equally distributed between five aquariums of 30 L each, previously conditioned to adequate water condition to the fish, with constant temperature of 28 °C using thermostat systems (Hopar Aquarium Heater H-606 150W, 5 ACS Paragon Plus Environment


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China), tap water was dechlorinated and pH adjusted to 6.50 (Seachem Prime water conditioner, Madison, GA, USA) and submitted to constant filtration and aeration (Aquathec FE25 Filtration System, China) with flow rate adjusted to 250 L/h. Fish were fed with commercial ration TetraMin (Tetra GmbH, Germany), adequate to tropical ornamental fish, twice a day (8 AM and 5 PM) and monitored during five days.

2.2. Preparation of composite particles

The alginate-chitosan particles were prepared by titration of alginate solution in chitosan solution under continuous stirring as a modified protocol from Ali et al.26 Typically, 1 mg/mL alginate solution was prepared by overnight stirring in the same aqueous buffer as chitosan (acetate buffer, 80 mM, pH 4.50 ± 0.02). The alginate solution (50 mL) was slowly titrated drop by drop at 2 mL/min in chitosan solution (1 mg/mL, 50 mL) under stirring (1200 rpm) and colloidal particles were obtained with the strong electrostatic interaction between the two macromolecules. After complete addition of alginate, the suspension was kept under stirring overnight. For preparation of drug containing particles, ivermectin or mebendazole, the drug was previously added to chitosan solution and the mixture was kept under overnight stirring (800 rpm). Next, the above protocol was followed. Particles of three different concentrations of each drug were prepared, namely 38, 15 and 4 µM. All particles dispersions were centrifuged (Sorvall Super T21 Centrifuge; Newtown, CT) at 21000 rpm, 4°C, during 20 min. The supernatant was removed, new equivalent volume of pure water (pH 6.3) was added and the samples were newly centrifuged during 20 min and the supernatant again removed. Following, all samples were lyophilized (Liotop Liobras, SP, Brazil). For DLS, zeta potential and SAXS experiments, at time of measurement 10 mg of particles were individually dispersed in 5 mL of water using a vortex mixer (Gehaka Av-2; SP, Brazil) for 10 s. An aliquot of 100 µL of each sample was diluted to 10 mL in different buffers for the studies of pH dependence. Buffer of pH 2.50 was prepared using sodium citrate and hydrochloric acid. Buffers of pH 3.79, 4.10, 4.32, 4.50 and 6.50 were all prepared with sodium acetate and acetic acid by varying proportions of salt and acid in order to achieve the final desired pH. Maximal variation in pH was 0.02. All buffers were at the same concentration of 80 mM. Chitosan and alginate solutions were also prepared at 0.1 mg/mL in five independent samples of each polymer for each pH for measurements of hydrodynamic diameter of the sole polymers. Solutions were stirred overnight (1500 rpm) at room temperature and the DLS analysis was performed right after.


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In order to evaluate eventual mass loss of components during particles preparation, triplicates of drug-free particles and drug-containing particles for the highest concentration of each drug were prepared as described above and the removed supernatants from centrifugations were also lyophilized. The recovered dried mass of the supernatants was weighted and subtracted from the initial mass in preparation and corresponded to an average of 16 mg. No expressive difference of recovered mass was found between samples comparing drug-free particles and drug-containing particles (see Supporting Information, Table S1), thus we assume that the recovered mass must correspond to alginate and/or chitosan. In order to identify the components of the washing process, the recovered mass was dispersed in 50 mL of phosphate buffer (pH 8.0, 80 mM) able to solubilize alginate. The mixture was stirred at 1500 rpm overnight at room temperature, than centrifuged as described above and supernatant was separated and saved. The pellet was submitted again to the same procedure and the new supernatant collected and added to the first one. Supernatants and pellet were lyophilized and the dry masses weighted corresponding to average 6 mg of the pellet and 9 mg of supernatant, considering an individual sample. The mass from the pellet was dissolved in 50 mL of buffer pH 2.5 and no precipitation was observed after centrifugation, hence corresponding to chitosan, while the other mass corresponded to alginate. Thereby, the correct concentrations of chitosan and alginate in final particles were 0.92 and 0.82 mg/mL, corresponding to 7 and 4 µM respectively.

2.3. Isothermal titration calorimetry

ITC measurements were performed with VP-ITC microcalorimeter from MicroCal Inc. (Northampton, MA). The working cell of 1.442 mL in volume was typically filled with a chitosan acetate buffer solution (pH 4.50 ± 0.02) free or containing mebendazole or ivermectin. The reference cell was filled with the same buffer. For ITC measurements the chitosan solution free of drug or containing the highest concentration of each drug was diluted to 0.5 µM of chitosan with the same buffer, while alginate was at 5 µM. One aliquot of 2 µL followed by 27 aliquots of 10 µL of alginate solution were injected stepwise with 200 s interval into the working cell. The corresponding reference blank experiments were also performed, namely titration of buffer in chitosan solution and titration of alginate solution in buffer. In order to avoid the presence of bubbles, all samples were degassed for 5 min shortly before starting the measurements. The sample cell was constantly stirred at a rate of 307 rpm, and the



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measurements were performed at 25 °C. The data analyses were carried out with Origin software provided by MicroCal.

2.4. Small angle X-ray scattering

SAXS data were obtained at the magnetic beamline SAS of the Brazilian Synchrotron Light Laboratory (LNLS). The composite particles samples were individually injected in a stainless-steel sample holder and temperature was stabilized at 25 °C. The wavelength of incident beam was 1.605 Å and the linear detector was placed at 43.5 cm from sample. Exposure time of 60 s was applied for intensity acquisitions. Resulting data were normalized to constant beam intensity and corrected for transmission, sample thickness, parasitic and background scattering, according to standard procedures.27 Intensity I was plotted as function of the wave vector q and curves were analyzed in the Porod’s region28, as discussed.

2.5. Dynamic light scattering

The DLS measurements of particles size and size distribution were performed with a Malvern Zetasizer 300 ZS (Malvern Instruments) operating with a 4 mW HeNe laser at a wavelength of 632.8 nm and detection at an angle of 173°. All measurements were performed in a temperature controlled chamber at 25 °C. The typical autocorrelation function was acquired using exponential spacing of the correlation time. The data analyses were performed with software provided by Malvern. The intensity weighted size distribution was obtained by fitting data with a discrete Laplace inversion routine.29 Size determination was made using Stokes-Einstein relation.30 The polydispersity of particles was accessed by using cumulant analysis of the correlation functions measured by DLS applying the amplitude of the correlation function and the relaxation frequency. The second-order cumulant was used to compute polydispersity of samples.31

2.6. Zeta potential

The zeta potential of particles was measured with the same Malvern Zetasizer performing at least 50 runs per sample at 25 °C. The principle of measurement is based on laser Doppler velocimetry. The mobility U is converted to zeta potential ζ using the Helmholtz


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Smoluchowski relation, ζ = Uη/εε0, where η is the solution viscosity, ε the dielectric constant of water, and ε0 the permittivity of free space.

2.7. In vivo experiments

Following the fifth day upon fish arrival and conditioning, ten specimens of Corydoras agassizii where randomly collected one by one from the aquariums using appropriate collecting net. The fish were euthanized in a benzocaine solution overdose (400 mg/L)32 in accordance with Brazilian law (Federal Law No. 11.794, dated 8 October 2008 and Federal Decree No. 6899, dated 15 July 2009), and all organs and body fluids were examined for parasites infections using an Axioplan 2 Zeiss microscope (Göttingen, Germany).33,34 For parasitological exams of stomachs and intestines, the organs were removed and placed in Petri dishes and then examined.35 For in vivo experiments larger amount of each particle was prepared as described in particles preparation and the suspensions were submitted to lyophilization (Liotop Liobras, SP, Brazil) in order to obtain the drug delivery structures in powder form. In this way oral administration is facilitated since the powder is easily combined with the normal commercial fish ration without changing physical properties of the later, besides stimulating particles intake. After lyophilization the samples were maintained in the fridge (4 °C) before use. At time of administration, a weighted amount of each particle was mixed with also weighted amount of ration and the fish were fed. The weighted amount of all administrated particles was similar between the aquariums, since drug-containing particles were previously prepared with different concentrations of the respective drug. The amount considered the drug dose, which was calculated in mg per kg of fish body weight considering the average weight of 4.83 g of the Corydoras fish. Starting at the seventh day after fish arrival, particles containing the three concentrations of ivermectin and drug-free particles were in this way administrated along the normal morning feeding (8 AM) to fish of four aquariums. The fifth aquarium was the control and fish were fed with ration alone. After the fifth, tenth and fifteenth day of administration, five specimens from each of the five aquariums were randomly collected and analyzed as described above. The same protocol was followed for mebendazole particles with no treated lot of fish and after renovation of water of the aquariums. The animal protocols to carry out during in vivo studies were approved by the Animal Ethical Committee of Federal University of Sao Paulo.



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2.8. Data fitting and calculations

The thermodynamic parameters of alginate binding to chitosan during in situ particles production were obtained from ITC by applying the model for a single set of identical binding sites using a nonlinear least-squares fitting.36,37 The model describes the equilibrium between unbound protonated chitosan monomers and unbound ionized alginate monomers. Hence, the binding constant K is defined as: ஀

‫ = ܭ‬ሺଵି஀ሻ஼

(1)

౜౨౛౛

where Θ = [bound alginate]/N[L] is the fraction of chitosan binding sites occupied by alginate monomers, [L] is the total concentration of chitosan protonated monomers, and the number of binding sites N represents the number of alginate monomers bound to each chitosan monomer at saturation of binding sites. In equation 1, Cfree is the concentration of free alginate representing: Cfree = Ctot − NΘ[L]

(2)

where Ctot is the total monomeric alginate concentration. The combination of equations 1 and 2 provides a quadratic equation for the molar fraction Θ, and by solving this equation one gets an expression for Θ as a function of chitosan concentration.37,38 Therefrom, the heat release per injection, δQ, where the bound fraction changes by δΘ, is δQ = N[L]V∆HδΘ, being V the volume in the sample cell and ∆H the molar enthalpy. By fitting the measured heat release δQ with this expression, the fitting parameters N, K, and ∆H are determined (for more details see 36-38

). Considering that chitosan characteristics in solution strongly depend on pH, degree of

protonation, DP%, was besides calculated considering respective buffers pH. In order to determine precise DP%, two approaches developed by Rinaudo et al. were applied.39,40 The first considers complete dissociation of acid, which applies for HCl buffer solution of pH 2.50. The degree of protonation is obtained via: pKa = pH + log10[α/(1 − α)]

(3)

where pKa=6 for chitosan39 and α is DP%. The second approach considers acetic acid equilibrium of dissociation and applies for acetate buffer solutions of pH 3.79, 4.10, 4.32, 4.50 and 6.50. One considers: α = (α”Ca − [H3O+])/Cp

(4)


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where Ca and Cp are concentrations of acid and polymer respectively, [H3O+] is calculated from pH of respective buffer solution and α” is degree of dissociation of weak acid in presence of chitosan: α” = Ka/(Ka + [H3O+])

(5)

where Ka is the dissociation constant calculated using pKa=4.75 (25 °C) for acetic acid.

3. RESULTS AND DISCUSSIONS

3.1. Thermodynamic characteristics and surface structure

The physical structure of drug delivery systems, which takes crucial part in effective in vivo performance aiming high treatment yields with low or inexistent side effects, is designed in preparation procedures relying on a variety of features such as type and concentration of components, solubilization procedures, conditions of components mixing, interaction between structuring molecules, etc. Herein, two biocompatible polysaccharides, namely alginate and chitosan (Figure 1), were employed in production of anti-parasitic drug delivery particles. Being strongly and oppositely ionized macromolecules in the pH range around 4.5041,42, both polymers establish electrostatic interaction leading to assembling of the final drug delivery particles as two main components composites. Hence, the intensity of interaction certainly designates the fate of this kind of particles in terms of its final physical structure. In order to quantitatively verify the assumption, we performed isothermal titration calorimetry (ITC) were the conditions of experiment were similar to the conditions of particles preparation procedure, i.e., alginate solution is slowly titrated in chitosan solution containing or not anti-parasitic drugs, under continuous stirring and constant pH and temperature. Figure 2a shows ITC results for interaction between alginate and chitosan in production of drug-free particles. The sharp down pointing peaks (upper panel) represent the heat released at every injection, and thus evidence strong exothermic effect when adding alginate solution to chitosan solution in constant pH 4.50. If comparing results of drug-free particles (Figure 2a) with ivermectin particles (Figure 2b) and mebendazole particles (Figure 2c), it is noticeable the reduction of heat released at every alginate injection into the calorimeter cell containing chitosan and drug. The integrated heats resulting from interaction as function of alginate/chitosan molar ratio (lower panels) show sigmoidal behavior, since with every following injection the heat signal decreases denoting partial neutralization of protonated chitosan with negatively charged alginate. At every following alginate injection there is less 11 ACS Paragon Plus Environment


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free chitosan available to interact and thus, the energy release with the next injection is also a little lower and so on. The heat release from the interaction ceases when all chitosan in the titration cell is complexed and neutralized with alginate; then, only heats of dilution are observed, which have been subtracted from the final thermodynamic analysis in this study, as described elsewhere.36,43 Overall, the strong heat released in titration is indicative for an energetically favorable interaction between the two macromolecules. Therefore, ITC measurements provide binding equilibrium constant and enthalpy variation due to electrostatic interaction during in situ particles preparation. In order to analyze the interaction, thermodynamic parameters were determined with ITC measurements employing the model for a single set of binding sites (see materials and methods) with a nonlinear least-squares fitting using Origin software. 36,43 This model describes the equilibrium between the free protonated chitosan monomers plus free ionized alginate monomers and the bound alginate/chitosan complex. The fitting of measured heat released actually produces the sigmoidal curves in Figure 2, and provides fitting parameters as the number of binding sites N, equilibrium constant K, and enthalpy variation ∆H. From K one calculates the Gibbs energy related to the interaction applying ∆G = −RT ln(55.55K), where ∆G is defined for standard state of mole fraction, T is temperature and 55.55 introduces concentration of water. Therefrom, the entropy gain is further estimated via T∆S = ∆H − ∆G. The fitting parameters along the calculated Gibbs energy and entropy gain are shown in Table 1. Accordingly, the number of binding sites N, which may be translated as number of ionized alginate monomers interacting with protonated chitosan monomers at saturation of binding sites, i.e., the point where no longer chitosan monomers are available to interact, is near 0.60 for drug-free particles and ivermectin particles. This value means 1/N ~ 1.7, which provides the ratio of accessible chitosan monomers to bind on alginate monomers at saturation. Intriguing, the ratio is closer to 2 chitosan monomers for 1 alginate monomer instead of 1:1 relation. As a matter of fact, in terms of monomers structure dimensions, for chitosan one estimates a monomer to monomer charge distance around 0.52 nm37 and for alginate the distance between adjacent bridge oxygens (see Figure 1a) is around 0.55 nm44, hence near dimensional features, which in a simplest interpretation would lead approximately to the 1:1 ratio at saturation. Moreover, in terms of macromolecular structure, there is an expressive difference in average molecular weight between alginate, 200 kDa, and chitosan, 130 kDa, meaning an amount of 1143 and 797 average monomers for each chain of the polymers respectively. In a hypothetic optimized situation one would expect one alginate chain


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interacting with one chitosan chain, which provides 1.43 alginate monomers to one chitosan monomer. Nevertheless, the experimental N ~ 0.60 evidences a far from ideal condition, which must be related to molecular conformations of both polymers in solution. Indeed, despite the similarity in basic chain geometry (cellulose type), chitosan is characterized as less extended than alginate.45 Hence, the higher flexibility of chitosan leads to conformation changes in solution producing worm-like structures.46 Thereby, the strong interaction with extended alginate leads to the proportion of ~1.7 chitosan monomers per alginate monomer at saturation. A higher difference was obtained for mebendazole particles, with N ~ 0.44 (Table 1), thus 1/N ~ 2.2. This result evidences that presence of mebendazole increased the amount of chitosan monomers per alginate if comparing to drug-free and ivermectin particles. On the other hand, the number of effective binding sites N was reduced meaning that a reduction in number of protonated chitosan monomers occurred, since binding sites depend on effective charge-charge interaction between COO- and NH3+. Presumably, the drug hydrophobic molecules of relative small structural dimension (Figure 1d) may be well distributed along the chitosan chains establishing hydrophobic interaction with the polymer backbone. Additionally, partial deprotonation of chitosan may occur when interacting with mebendazole considering competition of protons between chitosan amine and mebendazole ring nitrogens (pKa 6.6). Thus, the chitosan structural changes proportioned by mebendazole results in effective increase to ~2.2 chitosan monomers for one alginate monomer at saturation, but the effective number of binding sites N, i.e., interaction between the ion pair, decreased 27% in presence of mebendazole, relatively to the other two particles. Comparing to ivermectin, this later drug is of relative larger molecular dimension (Figure 1c), besides robust bulky structure, and one may speculate that drug-drug van der Waals and hydrophobic interactions possibly prevailed over drug-chitosan hydrophobic interaction, leading to lower distribution of individual ivermectin molecules along the polymer backbone and instead promoting localized drug aggregates, thereby contributing to N maintenance as for drug-free particles. The fitting function for heat release that is the sigmoidal curve (Figure 2) also provides binding constant K and molar enthalpy ∆H for particles production (Table 1). Now, the binding constant K is highly larger for drug-free particles comparing to ivermectin and mebendazole particles. Therefore, the binding strength is indeed higher in absence of drug, confirming strong electrostatic interaction between the two polysaccharides. The same conclusion accounts from molar enthalpy, where a significant negative ∆H in the order of thousand kcal/mol for drug-free particles, denotes intense exothermic effect during interaction. Nevertheless, both drug-



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containing particles still present highly negative ∆H evidencing that formation of all particles is thermodynamically driven and favored. The calculated Gibbs free energy ∆G is similar for the three particles (Table 1) indicating that despite ∆H differences between drug-free and drug-containing particles, all systems achieve a state of intense negative Gibbs energy, denoting that alginate-chitosan interaction effectively leads to particles formation, either in drugs presence. Furthermore, the entropy contribution T∆S is around two times highly negative for drug-free particles, despite also significantly negative for drug-containing particles. The large entropic decrease in formation of particles reinforces the strong interaction between the macromolecules, where free ionized and extended polysaccharides chains interact through electrostatic forces leading to assembling of composite particles. Noteworthy, heat contribution due to changes in alginate and chitosan conformation during interaction and assembling of the particles must further contribute to thermodynamic characteristics as discussed above. Especially, if considering drug-containing particles, the comparative less negative enthalpy variation observed must rely on reduced molecular flexibility of chitosan that is already bearing the drugs. Therefrom, the conditional increased stiffness of chitosan chains may reduce to some point the heat release in the complexation process. Other effects as hydrophobic interaction between polymers backbone, hydrogen bonding, deprotonation of chitosan amines, alteration in ions distribution, changes is water structure, may contribute some to heat release and some to heat absorption. Nevertheless, the still highly negative thermodynamic parameters obtained for all particles evidence overall the strong electrostatic interaction, which promotes the particles formation even in presence of large hydrophobic molecules of anti-parasitic drugs. The physical structure of the produced particles, discussed in detail in the next section, was preliminary evaluated in terms of surface aspect by means of SAXS. Figure 3 shows SAXS patterns of the same final particles discussed above. In the double logarithmic plot of scattering intensity (I) as function of scattering vector (q) it is evidenced that the curves follow Porod´s power-law in the region upward 0.1 nm-1, meaning that intensity I (q) is proportional to q-4 for larger values of q, as predicted for ideal spheres and surface fractals.28,47 The data also show a constant additive background resulting from threedimensional electron density fluctuations in higher q region. Of notice, I (q) curves do not present any distinct features; instead exhibit practically overlaid scattering patterns with constant slope in the Porod’s region. The simple assumption of Porod’s law for surface


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scattering I (q) ~ S q-4 denotes that surface area S for all the particles is constant, evidencing similar surface aspect for the same. The similarity in surface aspect of the particles further reinforces the observation that independently of drugs absence or presence of the two different anti-parasitic drugs studied herein, the particles are equally produced as a result of strong electrostatic forces leading to state of lower free energy thus with favored thermodynamic conditions, and providing drug delivery particles with same physical surface aspect.

3.2. Structural dependence on pH

Application of oral drug delivery micro or nanoparticles requires that these devices further ensure physical responses in various fluids of specific characteristics, which may allow stability during route of administration while permitting release of transported active compounds in targeted organs, e.g., gastrointestinal tract.48 During route of oral administration, pH varies drastically and hence the drug delivery particles may be developed in order to purposely respond to this variation. Therefore, our particles were submitted to a range of pH in buffer solutions in order to evaluate physical characteristics. Figure 4a shows DLS results for a set of composite chitosan-alginate particles as function of pH variation. Particles containing three concentrations of each drug, i.e., ivermectin or mebendazole, were analyzed along the control, drug-free particle. As shown, all particles presented strong pH dependence concerning hydrodynamic diameter. In pH 2.50, the particles show large average diameters on micrometer scale. Interestingly, when increasing pH to 3.79, size reduction is evidenced for all particles, but more abruptly to the drug-free particle. The same behavior is noticed in pH 4.10, were all particles assumed average size on nanometer scale. Similar behavior is shown in pH 4.32 with slight average size increase. In pH 4.50 average sizes show a new increment and finally in pH 6.50 all particles present submicrometric or micrometric diameters, but with smaller averages if compared to the initial pH 2.50 for the majority of samples. Polydispersity of all samples was large and in the order of 0.32 and 0.71 for all samples, indicating large size distribution of the composites. However, when analyzing the average diameters, size dependence on pH is evidenced and suggests structural peculiarities related to physical stability, ionization, protonation and hydration of the macromolecules. This assumption is corroborated by the behavior of sole alginate and sole chitosan in diluted solutions (0.1 mg/mL) of the various pHs, where hydrodynamic diameters of each polymer show strong dependence on pH (see Supporting Information, Table S2). 15 ACS Paragon Plus Environment


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Actually, sole chitosan presents reduced size in the pH range 2.50-4.50 denoting good solubility of the polymer chains, but aggregates in pH 6.50 as a result of deprotonation.38 Instead, alginate shows lower size in pH between 4.10-6.5, while aggregates in pH 2.5, evidencing formation of alginic acid of low water solubility.23 Additionally, zeta potential results depicted in Figure 4b show conformable tendency of particles structural dependence on pH. At pH 2.50, all particles present zeta potential around neutral to positive values, with the drug-free particle bearing the highest +22.3 mV on average. In pH 3.79 a slight decrease of zeta potential is noticed and similar tendency follows for pH 4.10, 4.32, 4.50 and 6.50. Actually, the drug-free particle shows an abrupt reversal from around +10 mV in pH 3.79 to around -21 mV in pH 4.10, plus decreasing to around -38 mV in pH 6.50. Samples containing mebendazole show approximately similar results, with nonsignificant drug concentration dependence. On the other hand, particles with invermectin have shown a relative discrete reduction of average zeta potential as function of pH increase, suggesting drug contribution to particles surface charge profile for the three studied concentrations. Maximal standard deviation for zeta potential was 4.3 mV and average conductivity was 6.38 mS/cm for all samples. As evidenced in Figure 4, both DLS and zeta potential results show that particles feature pH dependent characteristics. In strong acid condition the composites present larger size and neutral to positive zeta potential. These results may be related to physico-chemical properties of the macromolecules, especially to alginate. Indeed, alginate with pKa 3.38 (mannuronate M residues) and 3.65 (guluronate G residues), is in hydrated form in strong acid with protonation of carboxylic groups leading to alginic acid of low water solubility.23 Hydration of large chains of the polysaccharide leads to increase in hydration volume, which thus may increment hydrodynamic diameter of the particles producing swollen expanded structures, as observed and represented in Figure 5. Besides, chitosan remains ionized in pH 2.50 by complete protonation of amine groups (Table 2), hence leading to particles of positive surface charge, as determined. Since particles were prepared with same concentration of chitosan and alginate (w/w), the fact that zeta potential remains slightly positive or close to neutrality, instead of strongly positive, may suggest that chitosan is mostly overlaid by hydrated alginate or partially neutralized by the same. Indeed, the proper hydration of alginate must have contributed to particles partial neutralization. Thus, in absence of surface charge, aggregation of particles may also occur, besides contributing to size increase. In pH 3.79 all particles presented somewhat size reduction, but still relative large particles. In this condition pH is only slightly above the pKa of alginate guluronate residues, 16 ACS Paragon Plus Environment

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thus partial hydration of alginate keeps similar structure conditions as for strong acid medium, as discussed above. The protonation degree of chitosan is also not affected in this pH (Table 2). More pronounced changes were noticed in pH from 4.10. In fact, reasonable size reduction along charge reversal for the majority of samples was determined, indicating that this pH condition is ideal for production of particles of lower size. From pH 4,10 to 4.50 both alginate and chitosan are strongly ionized, i.e., alginate is negatively charged on carboxylic groups and chitosan is almost fully protonated on amine groups with very slight protonation degree reduction along this pH range (Table 2). Thereby, an ionized particle of lower size (Figure 5) is obtained and one may suggest the maintenance of strong electrostatic interaction between alginate and chitosan in the particle, as determined and discussed in the previous section. The fact that zeta potential is highly negative for the drug-free particle in the pH 4.10 to 4.50 range if comparing to the majority of drug containing particles, reinforces the assumption that alginate mostly overlays chitosan, and hence the negative charges of the former prevail on surface charge distribution. Additionally, the larger average molecular weight of alginate compared to chitosan with corresponding 1143 and 797 average monomers per chain as discussed before, further argue in the sense that extended chains of alginate may prevail over shorter chitosan in the complexation during particles assembly, leading to formation of structures mainly overlaid by alginate. In the final pH 6.50 a new size increment was observed for all samples along further reduction of zeta potential. In this condition, chitosan is predominantly neutralized, i.e., with low degree of protonation in the order of 21% (Table 2) while alginate remains strongly ionized. In absence of counter ions, now the negative charges of alginate develop intermolecular self-repulsion in the particles, which promotes bulk expansion leading to size increase as denoted and represented in Figure 5. Notwithstanding, since majority of zeta potential results are not very far from neutrality, some aggregation may occur in all samples. However, comparing Figure 4a and b, when zeta potential is closer to neutrality for a part of samples, e.g., samples containing ivermectin, the lower size was observed for the same in the corresponding pH. This further corroborates that the main process governing particles characteristics is in fact related to ionization and hydration of the macromolecules. The observation that ivermectin presence in the composites diminishes zeta potential variation as pH varies, comparing to mebendazole particles or drug-free particle, may be somewhat related to the large molecular structure of this drug, thus reducing effective surface charge of the particles. Besides the differences between average molecular weight of polymers, one has to take in account the order of components mixing in preparation of 17 ACS Paragon Plus Environment


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particles. The fact that drugs are previously mixed with chitosan, which is necessary to allow drug-chitosan association due to hydrophobic characteristic of drugs, increases the bulky structure of chitosan but without threatening the interaction with alginate as shown in ITC experiments. Following, the titration of alginate solution in chitosan-drug solution leads to particles with predominance of alginate over surface and chitosan-drug in the core, since SAXS suggests the same physical surface aspect for all particles, independently of drug absence or kind of drug. Finally, zeta potential of ivermectin particles is less influenced with pH variation further suggesting a scenario where chitosan is more internalized in the particle with the bulky drug, and thus ionized carboxylic groups of alginate tend to face the core of particles in order to interact with chitosan protonated amines. This scenario would lead to particles of effective weak surface charge in the various pH ranges as determined for ivermectin particles. Actually, this particle profile where the drug is predominantly localized in the particle core, may account for protection during storage and route of administration, e.g., protecting the drug from undesired degradation, and can furthermore alter pharmacokinetics and pharmacodynamics of the drug delivery.

3.3. In vivo performance

Aiming to evaluate practical performance of the drug delivery composite particles, we performed in vivo experiments in where the particles were systematically administrated via oral to Corydoras agassizii fish collected from the Amazon basin (Figure 6a). Before the experiments, the sampled fish were analyzed after few days upon arrival, as detailed in the experimental section, and they were highly infected with nematodes (genus Procamallanus) and digenetic trematodes (Figure 6b) in the whole digestive tract. The parasites were found in adult stage for nematode and metacercariae for digenetic trematode and prevalence was 100% for both (Figures 7a and c). No other kind of parasites was found by examining the fishes whole body. The experiments consisted in daily feeding the fish with lyophilized particles, once a day together with the morning normal feeding. In the first experiment ivermectin particles with three concentrations of drug were individually administrated to fish of three aquariums and drug-free particles to fish in the fourth aquarium. The fish of the fifth aquarium were fed without particles as a control (see experimental section for details). Sampled fish from each aquarium were analyzed at the fifth, tenth and fifteenth day of treatment and the analysis focused in nemotodes treatment, since ivermectin is ineffective against trematodes due the absence of high-affinity binding sites.49 As 18 ACS Paragon Plus Environment

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shown in Figure 7a, parasites prevalence remained at 100% equally for the control and drugfree particles during whole experiment. This result evidenced that drug-free particles itself do not provide treatment, as expected. Besides, the same drug-free particles did not alter any visible physical aspect or behavior of the fish, comparing to the control. Regardless, ivermectin particles showed systematic reduction of parasites prevalence: the lower concentration (0.1 mg/kg) provided decrease to 80% (10th day) and to 40% (15th day); the intermediate concentration (0.2 mg/kg) provided decrease to 60% (10th day) and to 0% (15th day); while the higher concentration (0.4 mg/kg) reduced to 80% (5th day), 20% (10th day) and to 0% (15th day). However, the higher concentration promoted 20% mortality and the intermediate concentration 5% mortality during the course of study, while no mortality was observed for the lower concentration (Figure 7b). For the higher concentration, lethargy and darkening of fish color was also observed. These results are not surprising considering the known relative neurotoxicity of ivermectin.50,51 Indeed, previous studies in Atlantic salmon, where 0.5 mg/kg of ivermectin was administrated every other day sprayed directly over food, reported high mortality over 85% during 15 days of treatment.52 In our study the mortality was lower for the higher concentration during the same period, which suggests improvement of treatment via drug delivery particles. Moreover, despite oral administration being the most viable route of fish treatment,53 one has to consider the fact that distribution of actives in feeding is not proportional between fish, i.e., some may ingest more particles than others, which may have contributed to mortality observed for the higher concentration of ivermectin. Actually, nematodes treatment was globally more efficient with 0.2 mg/kg ivermectin particles for which low mortality was observed, besides complete elimination of parasites in 15 days. The second experiment was conducted in the same conditions using mebendazole particles and focused in trematodes treatment. Figure 7c equally shows 100% prevalence for the control and drug-free particles during 15 days, as observed in the first experiment, confirming that particles alone have any effect over fish and parasites. Now, the particle of lower concentration of drug (0.1 mg/kg) provided reduction to 80% prevalence only at the 15th day; with intermediate concentration (0.2 mg/kg) reduction to 80% was achieved at the 10th day and 60% at the 15th day; the higher concentration (0.4 mg/kg) provided the better reduction profile to 80, 60 and 40% respectively at 5th, 10th and 15th day. Of notice, the experiment employing mebendazole particles did not cause mortality nor lethargy, neither physical aspect and behavior changes, confirming good safety of the drug as described elsewhere.54 These later results may suggest that concentrations of mebendazole where insufficient to completely eliminate the parasites. However, one has to further consider that digenetic 19 ACS Paragon Plus Environment


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trematodes are of difficult treatment especially because this class of parasites is generally absent in γ-amino-butyric acid (GABA), which is a main target of antiparasitic drugs, leading to flaccid paralysis of parasites.55 Mebendazole also acts binding on β-tubulin molecule, thereby disrupting cell functions such as cell division and transport, which hence may be effective on digenetic trematodes.55 Despite, low treatment yields have been described so far.56 As a matter of fact, efficacy of treatment obviously depends on efficacy of the drug action. However, the use of particles provides better vectorization, i.e., optimized delivery of the drug to the site of expected action with reduced route degradation. Thereby, results of these experiments evidence that the composite particles allowed treatment and showed enhanced yields comparing to previous studies, suggesting that the composites may be applied as drug delivery system. Moreover, this is the first in vivo study performed in ornamental fish from the Amazon basin applying composite drug delivery particles for treatment of endoparasites. Hence, direct comparison with results on fish from other species cannot be made. Nevertheless, one emphasizes the importance of treating ornamental fish designed for exportation in the sense that international translocation of parasites may represent a serious concern.57 Therefore, if parasites spread and develop in new environments it may represent a catastrophic environmental menace, with outbreak of pathologies and disturbance of natural ecosystems.57-59 Importantly, the analyzed Corydoras fish presented high prevalence of 100% for both endoparasites, which by turn allowed proper evaluation of the particles as drug delivery devices for improved treatment of the fish gastrointestinal parasites. The experiments were conducted during 15 days in order to optimize fish treatment in a relative short period, aiming to avoid fish suffering and taking in account demands of ornamental fish industry that requires fast treatments, thereby reducing long periods of fish storage, which may increase costs and mortality thus leading the trade unprofitable. The number of fish submitted to the experiments was also optimized in order to minimize fish harm and avoid many and unnecessary necropsy, following animal etic procedures. In terms of particles advantage, the proper composition of particles, made with alginate and chitosan, may have contributed to ingestion of particles since both polysaccharides are components of fish nourishment, e.g., used in production of commercial fish food for increase of palatability and nutrition yields.60 In terms of particles performance, as characterized in the previous section, when placed in the aquarium water of pH around 6.5, the same get negative surface charge which may provide physical stability during route of administration. Indeed, when ingested by the fish, particles initially face the buccopharyngeal membranes where pH ranges around water pH and thus the negative surface charge of particles is crucial to reduce 20 ACS Paragon Plus Environment

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mucous interaction in these regions, considering the electrostatic repulsion to the negative surface charge of cells.61 Hence, this may allow transit of particles and low retention. Thereby, particles achieve stomach where pH varies around 2.5 in fasting,62 which promotes charge reversal to particles. Now with positive surface charge, particles may interact with stomach mucosa of strong negative charge due the presence of transmembrane mucin glycoproteins.63 This scenario may allow prolonged permanence of the particles in stomach. Also during digestion of food, pH can increase until around 4.5.62 Hence pH variations and the proper natural digestion process induce particles slow degradation with attenuated release of drugs to the gastrointestinal tract, promoting the final parasites treatment. This ideal performance is certainly not restrictive to fish parasites treatment, but may be applied to a plethora of finalities in living beings, yet human application, denoting the composite particles as promising drug delivery system.

4. CONCLUDING REMARKS

The production, structure characteristics and in vivo performance of pH-responsive biocompatible drug delivery particles is described. Intense electrostatic interaction between ionized alginate and protonated chitosan promotes assembling of particles under thermodynamically favored conditions. Inclusion of model drugs of small or large molecular structure do not impede the particles production neither spoil their structural main features. The final multifunctionality of the particles relies on structural changes and surface charge changes, which concomitantly respond to pH variation. Hydration of large chains of the alginate polysaccharide leads to increase in hydration volume, which thus increments hydrodynamic diameter of the particles producing swollen expanded structures in pH 2.50. The further ionization of alginate in pH between 4.10 and 4.50 leads to charge reversal and compaction of particles under strong interaction with protonated chitosan. Beyond, deprotonation of chitosan in pH 6.50 prompts ionized alginate self-repulsion producing new swelling of the particles structure. The particles are thus pH structure responsive in the sense of swelling up or compress at the same time surface charge adequates to pH condition, which may generate advantageous physical stability and charge antagonism with cells during route of administration, while interaction activity may be induced at the target of the intended drug delivery. The efficacy of the particles was proven with incorporation of two model anti-parasitic drugs and in vivo application as oral administration in highly parasitized Corydoras fish. The in vivo performance denotes the effective action of the particles in treatment of fish parasites 21 ACS Paragon Plus Environment


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infection, highlighting the pH responsive devices as prominent biomaterial of real application in biological, biomedical an environmental fields.

SUPPORTING INFORMATION Data relative to mass lost in particles preparation (Table S1) and DLS data for chitosan and alginate (Table S2). This material is available free of charge via Internet at http://pubs.acs.org.

ACKNOWLEDGEMENTS The authors thank Brazilian Synchrotron Light Laboratory (LNLS) for allowing SAXS experiments and Prof. Maria A. Juliano for providing and supporting lyophilization of samples. The study was supported by Sao Paulo Research Foundation, FAPESP, through research grants: 2015/23948-5 and 2016/13368-4. A.C.M.F.P. also thanks FAPESP for MSc fellowship (process: 2017/16722-6).

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(28) Stoeckel, D.; Wallacher, D.; Zickler, G. A.; Perlich, J.; Tallarek, U.; Smarsly, B. M. Coherent analysis of disordered mesoporous adsorbents using small angle X-ray scattering and physisorption experiments. Phys. Chem. Chem. Phys. 2014, 16, 6583-6592. (29) Souza, C. F. V.; Faccin, D. J. L.; Mertins, O.; Heck, J. X.; Silveira, N. P.; Secchi, A. R.; Ayub, M. A. Z. Kinetics of thermal inactivation of transglutaminase from a newly isolated Bacillus circulans BL32. J. Chem. Technol. Biotechnol. 2009, 84, 1567-1575. (30) Mertins, O. Estudos físico-químicos e estruturais de lipossomas compósitos de fosfatidilcolina e quitosana. Ph.D. Thesis. Universidade Federal do Rio Grande do Sul: Brazil, 2008, http://hdl.handle.net/10183/14354. (31) Berne, B. J.; Pecora, R. Dynamic Light Scattering: with applications to Chemistry, Biology, and Physics. Dover Publications: New York, 2000, 376p. (32) Mathews, P. D.; Silva, M. R. M.; Maia, A. A. M; Adriano, E. A. Ultrastructure and ssrRNA sequencing of Myxidium amazonense n. sp. a myxosporean parasite of Corydoras melini from the Rio Negro river, Amazonas state, Brazil. Parasitol. Res. 2015, 114, 4675-4683. (33) Espinoza, L. L.; Mertins, O.; Gama, G. S.; Patta, A. C. M. F.; Mathews, P. D. A new Myxidium species (Myxozoa: Myxosporea) infecting the gallbladder of the turtle Podocnemis unifilis (Testudines: Podocnemididae) from Peruvian Amazon. Acta Trop. 2017, 172, 75-79. (34) Vázquez, N. D.; Mathews, P. D.; Chu-Koo, F. W.; Martín, S. T.; Orbe, R. I. Fauna parasitaria de juveniles de arahuana, Osteoglossum bicirrhosum (Vandelli, 1829) cultivado en el Centro de Investigaciones de Quistocoha, Loreto, Perú. Folia Amazonica 2007, 16, 29-33. (35) Mathews, P. D.; Mathews, J. P. D.; Orbe, R. I. Parasitic infections in juveniles of Prochilodus nigricans kept in semi-intensive fish farm in the Peruvian Amazon. Bull. Eur. Ass. Fish Pathol. 2013, 33, 28-32. (36) Mertins, O.; Dimova, R. Binding of chitosan to phospholipid vesicles studied with isothermal titration calorimetry. Langmuir 2011, 27, 5506-5515. (37) Ma, P. L.; Lavertu, M.; Winnik, F. M.; Buschmann, M. D. New insights into chitosan DNA interactions using isothermal titration microcalorimetry. Biomacromolecules 2009, 10, 1490-1499.



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(38) Mertins, O.; Dimova, R. Insights on the interactions of chitosan with phospholipid vesicles. Part I: effect of polymer deprotonation. Langmuir 2013, 29, 14545-14551. (39) Rinaudo, M.; Pavlov, G.; Desbrieres, J. Solubilization of chitosan in strong acid medium. Int. J. Polym. Anal. Charact. 1999, 5, 267-276. (40) Rinaudo, M.; Pavlov, G.; Desbrieres, J. Influence of acetic acid concentration on the solubilization of chitosan. Polymer 1999, 40, 7029-7032. (41) Mertins, O.; Schneider, P. H.; Pohlmann, A. R.; Silveira, N. P. Interaction between phospholipids bilayer and chitosan in liposomes investigated by 31P NMR spectroscopy. Colloids Surf., B: Biointerfaces 2010, 75, 294-299. (42) Baños, F. G. D.; Peña, A. I. D.; Cifre, J. G. H.; Martínez, M. C. L.; Ortega, A.; Torre, J. G. Influence of ionic strength on the flexibility of alginate studied by size exclusion chromatography. Carbohydr. Polym. 2014, 102, 223-230. (43) ITC data analysis in Origin. In Tutorial Guide, 7th ed.; MicroCal LLC: Northampton, MA, 2004. (44) Smidsrod, O.; Glover, R. M.; Whittington, S. G. The relative extension of alginates having different chemical composition. Carbohydr. Res. 1973, 27, 107-118. (45) Christensen, B. E.; Vold, I. M. N.; Vårum, K. M. Chain stiffness and extension of chitosans and periodate oxidised chitosans studied by size-exclusion chromatography combined with light scattering and viscosity detectors. Carbohydr. Polym. 2008, 74, 559-565. (46) Schatz, C.; Viton, C.; Delair, T.; Pichot, C.; Domard, A. Typical physicochemical behaviors of chitosan in aqueous solution. Biomacromolecules 2003, 4, 641-648. (47) Porod, G. In Small Angle X-ray Scattering. Glatter, O.; Kratky, O., Eds.; Academic Press: London, 1982; pp. 17-51. (48) Surnar, B.; Jayakannan, M. Stimuli-responsive poly(caprolactone) vesicles for dual drug delivery under the gastrointestinal tract. Biomacromolecules 2013, 14, 4377-4387. (49) Neal, M. J. Medical pharmacology at a glance, 8th ed.; John Wiley & Sons, Ltd., 2016. (50) Høy, T.; Horsberg, T. E.; Nafstad, I. The disposition of ivermectin in Atlantic salmon (Salmo salar). Pharmacol. Toxicol. 1990, 67, 307-312. 26 ACS Paragon Plus Environment

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(51) Horsberg, T. E. Avermectin use in aquaculture. Curr. Pharm. Biotechnol. 2012, 13, 10951102. (52) Johnson, S. C.; Kent, M. L.; Whitaker, D. J.; Margolis, L. Toxicity and pathological effects of orally administered ivermectin in Atlantic, chinook, and coho salmon and steelhead trout. Dis. Aquat. Org. 1993, 17, 107-112. (53) Ji, J.; Torrealba, D.; Ruyra, A.; Roher, N. Nanodelivery systems as new tools for immunostimulant or vaccine administration: targeting the fish immune system. Biology 2015, 4, 664-696. (54) Chagas, E. C.; Araújo, L. D.; Martins, M. L.; Gomes, L. C.; Malta, J. C. O.; Varella, A. B.; Jerônimo,

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dietary

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(Platyhelminthes: Dactylogyridae) and does not alter the physiology of the freshwater fish Colossoma macropomum (Cuvier, 1818). Aquaculture 2016, 464, 185-189. (55) Martin, R. J. Modes of action of anthelmintic drugs. Vet. J. 1997, 154, 11-34. (56) Reimschuessel, R.; Gieseker, C.; Poynton, S. In vitro effect of seven antiparasitics on Acolpenteron ureteroecetes (Dactylogyridae) from largemouth bass Micropterus salmoides (Centrarchidae). Dis. Aquat. Org. 2011, 94, 59-72. (57) Whittington, R.; Chong, R. Global trade in ornamental fish from an Australian perspective: the case for revised import risk analysis and management strategies. Prev. Vet. Med. 2007, 81, 92-116. (58) Camus, A. C.; Dill, J. A.; McDermott, A. J.; Clauss, T. M.; Berliner, A. L.; Boylan, S. M.; Soto, E. Francisella noatunensis subsp. orientalis infection in Indo-Pacific reef fish entering the United States through the ornamental fish trade. J. Fish Dis. 2013, 36, 681-684. (59) Camus, A. C.; Dill, J. A.; Rosser, T. G.; Pote, L. M.; Griffin, M. J. Myxobolus axelrodi n. sp. (Myxosporea: Myxobolidae) a parasite infecting the brain and retinas of the cardinal tetra Paracheirodon axelrodi (Teleostei: Characidae). Parasitol. Res. 2017, 116, 387-397. (60) Volpe, M. G.; Varricchio, E.; Coccia, E.; Santagata, G.; Di Stasio, M.; Malinconico, M.; Paolucci, M. Manufacturing pellets with different binders: effect on water stability and feeding response in juvenile Cherax albidus. Aquaculture 2012, 324-325, 104-110.



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Legends Table 1. Effective thermodynamic parameters (number of binding sites, N; binding constant, K; molar enthalpy, ∆H; Gibbs energy, ∆G; entropic contribution, T∆S) characterizing the interaction between alginate and chitosan for drug-free particles (ALGCH) and respective drugcontaining particles at 25 °C, as indicated.

Table 2. Calculated percentage of degree of protonation (DP%) for chitosan amine groups in a range of pH for different buffer solutions.

Figure 1. Molecular structures of ionized alginate containing m guluronic units and n mannuronic units (a), idealized chitosan (b), ivermectin (c) and mebendazole (d).

Figure 2. ITC data for the titration of alginate solution (ALG, 5 µM) into chitosan solution (CH, 0.5 µM), both in a 80 mM acetate buffer at pH 4.50 (25 °C). The heat flow (upper panels) and the respective integrated binding heats (lower panels) are plotted for particles free of drug (a), particles containing 2.5 µM ivermectin (b) and particles containing 2.5 µM mebendazole (c). The solid sigmoidal curves in lower panels are fits according to the model for one set of binding sites (see text for details and Table 1 for the corresponding fitting parameters).

Figure 3. Double-logarithmic plot of the SAXS patterns as intensity I as function of scattering vector q for the drug-free particles (ALGCH), ivermectin-containing particles (ALGCH-IVM) and mebendazole-containing particles (ALGCH-MB). Shifted curves slope (solid line) is shown for the Porod’s region.


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Biomacromolecules

Figure 4. Hydrodynamic diameters (a) and zeta potential (b) as function of pH for drug-free particles (); particles containing 4 (●), 15 (▼) and 38 µM (▲) of ivermectin; and particles containing 4 (◄), 15 () and 38 µM (►) of mebendazole. Solid lines are guide to the eye.

Figure 5. Representation of alginate-chitosan composite particles. In strong acid solution of pH 2.5-3.8 alginate chains (blue) are neutralized by hydrolysis while chitosan chains (red) present high degree of protonated amine groups, thus positive charges; these conditions produce a swollen expanded and positively charged structure. In mild acid solution of pH 4.1-4.5 both alginate and chitosan are oppose charged and strong electrostatic interaction produces a compact size reduced structure. In slight acid solution of pH 6.5 chitosan is neutralized, but highly ionized alginate leads to swelling by anionic self-repulsion producing a larger and negatively charged structure.

Figure 6. Snapshot of a Corydoras agassizii fish (a) collected from the Amazon basin and an isolated digenetic trematode (b) found in the fish digestive tract (scale bar spans 500 µm).

Figure 7. Prevalence of gastrointestinal nematodes in Corydoras agassizii fish during 15 days treatment using ivermectin-containing alginate-chitosan composite particles with 0 (), 0.1 (▼), 0.2 (▲) and 0.4 (●) mg of ivermectin per kg of average fish body weight (a). Percentage of fish mortality occasioned by ivermectin concentrations during parasitosis treatment (b). Prevalence of gastrointestinal digenetic trematodes in Corydoras agassizii fish during 15 days treatment using mebendazole-containing alginate-chitosan composite particles with 0 (), 0.1 (▼), 0.2 (▲) and 0.4 (●) mg of mebendazole per kg of average fish body weight (c).



Page 30 of 35

Table 1.

Particle

N

K

∆H

∆G

T∆ ∆S

(10 M )

(kcal/mol)

(kcal/mol)

(kcal/mol)

8

-1

ALGCH

0.608 ± 0.003

24.9 ± 7.24

-1077 ± 11

-15.21 ± 0.18

-1061

ALGCH Ivermectin

0.603 ± 0.014

1.90 ± 0.65

-615 ± 21

-13.68 ± 0.85

-601

ALGCH Mebendazole

0.441 ± 0.011

3.52 ± 1.42

-14.05 ± 0.89

-427

-718 ± 26

Table 2.

pH

2.50

3.79

4.10

4.32

4.50

6.50

DP (%)

100

100

98.8

97.9

96.9

21.3


Page 31 of 35

Figure 1.

a

c

b d

Figure 2.

b 150

0 -200 -400 -600 -800 -1000 -1200 0.0

0.5

1.0

1.5

ALG/CH (molar ratio)

2.0

c

Time (min) 0.2

0

50

100

150

0.0 -0.2 -0.4 -0.6 -0.8 -1.0 0 -100 -200 -300 -400 -500 -600 -700 0.0

0.5

1.0

1.5


2.0

Time (min) 0.2

Heat rate (µcal/sec)

100


0.2 0.0 -0.2 -0.4 -0.6 -0.8 -1.0 -1.2 -1.4 -1.6

50

Q (kcal/mol of injectant)

Time (min) 0



a


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

0

50

100

0.0 -0.2 -0.4 -0.6 -0.8 -1.0 -1.2 0 -100 -200 -300 -400 -500 -600 -700 -800 0.0

0.5

1.0

1.5



150

2.0

Biomacromolecules

Figure 3.

6

10

log I (a.u.)

, Porod s law

ALGCH ALGCH-IVM ALGCH-MB

5

10

0.1

0.2

0.3

0.4 0.5

5

6

7

5

6

7

log q (nm-1)

Figure 4.

Hydrodynamic diameter (nm)

a

1600 1400 1200 1000 800 600 400 200 2

3

4

pH

b 20 10

Zeta potential (mV)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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0 -10 -20 -30 -40 2

3

4

pH


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Figure 5.

Figure 6.

a

b


Biomacromolecules

Figure 7.

a 100

Prevalence (%)

80 60 40 20 0 0

5

10

15

days

b

Fish (%)

20

10

0

0.4

0.1

0.2

Ivermectin (mg/kg)

c 100

Prevalence (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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80 60 40 20 0 0

5

10

15

days


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Biomacromolecules

Table of Contents only graphic:


Targeted Drug Delivery and Treatment of Endoparasites with

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